Perhydrolase providing improved peracid stability

ABSTRACT

An acetyl xylan esterase variant having perhydrolytic activity is provided for producing peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen. More specifically, a  Thermotoga maritima  acetyl xylan esterase gene was modified using error-prone PCR and site-directed mutagenesis to create an enzyme catalyst characterized by an increase in the ratio of peracetic acid formation to peracetic acid hydrolysis specific activities (PAAF/PAAH ratio). The variant acetyl xylan esterase may be used to produce peroxycarboxylic acids suitable for use in a variety of applications such as cleaning, disinfecting, sanitizing, bleaching, wood pulp processing, and paper pulp processing applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/632,438 filed Dec. 7, 2009, now pending, which is incorporated byreference in its entirety.

TECHNICAL FIELD

The invention relates to the field of peroxycarboxylic acid biosynthesisand enzyme catalysis. More specifically, an enzyme catalyst comprising avariant enzyme having perhydrolytic activity is provided having anincrease in the ratio of peracetic acid formation (PAAF) specificactivity relative to peracetic acid hydrolysis (PAAH) specific activity(PAAF/PAAH ratio) when compared to the PAAF/PAAH ratio of the Thermotogamaritima wild-type perhydrolase. Use of the variant enzyme enhances theyield and stability of the peracid produced by enzymatic perhydrolysis.Methods of using the present enzyme catalyst to produce peroxycarboxylicacids are also provided.

BACKGROUND

Peroxycarboxylic acid compositions can be effective antimicrobialagents. Methods of using peroxycarboxylic acids to clean, disinfect,and/or sanitize hard surfaces, textiles, meat products, living planttissues, and medical devices against undesirable microbial growth havebeen described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S.Pat. No. 6,518,307; U.S. Patent Application Publication No.2003-0026846; and U.S. Pat. No. 5,683,724). Peroxycarboxylic acids havealso been used in a various bleaching applications including, but notlimited to, wood pulp bleaching/delignification and laundry careapplications (European Patent 1040222B1; U.S. Pat. No. 5,552,018; U.S.Pat. No. 3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No.5,364,554). The desired efficacious concentration of peroxycarboxylicacid may vary according to the product application (for example, ca. 500ppm to 1000 ppm for medical instrument disinfection, ca 30 ppm to 80 ppmfor laundry bleaching or disinfection applications) in 1 min to 5 minreaction time at neutral pH.

Enzymes structurally classified as members of family 7 of thecarbohydrate esterases (CE-7) have been employed as perhydrolases tocatalyze the reaction of hydrogen peroxide (or alternative peroxidereagent) with alkyl esters of carboxylic acids in water at a basic toacidic pH range (from ca. pH 10 to ca. pH 5) to produce an efficaciousconcentration of a peroxycarboxylic acid for such applications asdisinfection (such as medical instruments, hard surfaces, textiles),bleaching (such as wood pulp or paper pulp processing/delignification,textile bleaching and laundry care applications), and other laundry careapplications such as destaining, deodorizing, and sanitization(Published U.S. Patent Application Nos. 2008/0176783, 2008/0176299,2009/0005590, and 2010/0041752 to DiCosimo et al.). The CE-7 enzymeshave been found to have high specific activity for perhydrolysis ofesters, particularly acetyl esters of alcohols, diols and glycerols.However, CE-7 perhydrolases may also hydrolyze the carboxylic acid estersubstrate. As such, it is often preferable to employ an enzyme catalysthaving high selectivity for perhydrolysis (P) relative to hydrolysis (H)when synthesizing peroxycarboxylic acids from carboxylic acid esters(i.e., an enzyme catalyst having a higher “P to H” ratio). PublishedU.S. Patent Application No. 2010/0087529 to DiCosimo et al. describesseveral variant CE-7 perhydrolases derived from several Thermotoga sp.having higher perhydrolytic specific activity and/or improvedselectivity for perhydrolysis when used to prepare peroxycarboxylic acidfrom carboxylic acid esters.

Although the CE-7 family of carbohydrate esterases has been identifiedas a class of perhydrolytic enzymes having desirable specific activitiesfor peroxycarboxylic acid formation (e.g., peracetic acid formation;PAAF) and/or desirable perhydrolysis to hydrolysis (P/H) ratios forcarboxylic acid ester substrates, these enzymes may also have anundesirable enzymatic activity for hydrolyzing the peroxycarboxylic acidproduct (e.g., peracetic acid hydrolysis; PAAH) to the correspondingcarboxylic acid and hydrogen peroxide. As such, an enzyme catalystcomprising a CE-7 perhydrolase characterized by a higher PAAF/PAAH ratiomay provide greater peroxycarboxylic acid stability in formulationscomprising the enzyme catalyst.

The problem to be solved is to provide an enzyme catalyst comprising aCE-7 carbohydrate esterase having perhydrolytic activity and a higherPAAF/PAAH ratio of specific activities.

SUMMARY

A nucleic acid molecule encoding the Thermotoga maritima acetyl xylanesterase (SEQ ID NO: 2) was mutated by error-prone PCR and/orsite-directed mutagenesis to create a library of variant perhydrolases.Several perhydrolase variants were identified exhibiting an increase inthe ratio of peracetic acid formation (PAAF) to peracetic acidhydrolysis (PAAH) specific activities when compared to the PAAF/PAAHratio of the wild-type Thermotoga maritima perhydrolase having aminoacid sequence SEQ ID NO: 2 under the same assay conditions.

In one embodiment, an isolated nucleic acid molecule encoding apolypeptide having perhydrolytic activity is provided selected from thegroup consisting of:

-   -   (a) a polynucleotide encoding a polypeptide having perhydrolytic        activity, said polypeptide comprising the amino acid sequence of        SEQ ID NO: 26;    -   (b) a polynucleotide comprising the nucleic acid sequence of SEQ        ID NO: 25; and    -   (c) a polynucleotide fully complementary to the polynucleotide        of (a) or (b).

In other embodiments, a vector, a recombinant DNA construct, and arecombinant host cell comprising the present polynucleotide are alsoprovided.

In another embodiment, a method for transforming a cell is providedcomprising transforming a cell with the above nucleic acid molecule.

In another embodiment, an isolated polypeptide having perhydrolysisactivity is provided comprising amino acid sequence SEQ ID NO: 26.

In one embodiment, the variant polypeptide having perhydrolytic activityis characterized by at least a 1.1-fold increase in the PAAF/PAAH ratioof specific activities when compared to the PAAF/PAAF ratio of specificactivities of the Thermotoga maritima wild-type sequence SEQ ID NO: 2.

In another embodiment, a process for producing a peroxycarboxylic acidis also provided comprising:

-   -   (a) providing a set of reaction components comprising:        -   (1) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆=C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆=C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety optionally substituted with                    hydroxyl groups; wherein each carbon atom in R₅                    individually comprises no more than one hydroxyl                    group or no more than one ester group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=1 to the number of carbon atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁=C1 to C7 straight chain or branched chain                    alkyl optionally substituted with an hydroxyl or a                    C1 to C4 alkoxy group and R₃ and R₄ are individually                    H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acetylated monosaccharides, acetylated                disaccharides, or acetylated polysaccharides; and

            -   (v) any combination of (i) through (iv);

        -   (2) a source of peroxygen; and

        -   (3) an enzyme catalyst comprising the polypeptide of claim            5;

    -   (b) combining the set of reaction components under suitable        reaction conditions whereby peroxycarboxylic acid is produced;        and

    -   (c) optionally diluting the peroxycarboxylic acid produced in        step (b).

In another embodiment, a process is provided further comprising a step(d) wherein the peroxycarboxylic acid produced in step (b) or step (c)is contacted with a hard surface, an article of clothing or an inanimateobject whereby the hard surface, article of clothing or inanimate objectis disinfected, sanitized, bleached, destained, deodorized or anycombination thereof.

In another embodiment, a composition is provided comprising:

-   -   (a) a set of reaction components comprising:        -   (1) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆=C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆=C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety optionally substituted with                    hydroxyl groups; wherein each carbon atom in R₅                    individually comprises no more than one hydroxyl                    group or no more than one ester group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=1 to the number of carbon atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁=C1 to C7 straight chain or branched chain                    alkyl optionally substituted with an hydroxyl or a                    C1 to C4 alkoxy group and R₃ and R₄ are individually                    H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acetylated monosaccharides, acetylated                disaccharides, or acetylated polysaccharides; and

            -   (v) any combination of (i) through (iv);

        -   (2) a source of peroxygen; and

        -   (3) an enzyme catalyst comprising the polypeptide of claim            5; and

    -   (b) at least one peroxycarboxylic acid formed upon combining the        set of reaction components of (a).

The present process produces the desired peroxycarboxylic acid uponcombining the reaction components. The reaction components may remainseparated until use.

In a further aspect, a peracid generation and delivery system isprovided comprising:

-   -   (a) a first compartment comprising        -   (1) an enzyme catalyst comprising the polypeptide of claim            5;        -   (2) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆=C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆=C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety optionally substituted with                    hydroxyl groups; wherein each carbon atom in R₅                    individually comprises no more than one hydroxyl                    group or no more than one ester group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=1 to the number of carbon atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁=C1 to C7 straight chain or branched chain                    alkyl optionally substituted with an hydroxyl or a                    C1 to C4 alkoxy group and R₃ and R₄ are individually                    H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acetylated monosaccharides, acetylated                disaccharides, or acetylated polysaccharides; and

            -   (v) any combination of (i) through (iv); and

        -   (3) an optional buffer; and

    -   (b) a second compartment comprising        -   (1) source of peroxygen;        -   (2) a peroxide stabilizer; and        -   (3) an optional buffer.

In a further embodiment, a laundry care composition is providedcomprising a polypeptide comprising amino acid sequence SEQ ID NO: 26.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions.

The symbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleic acid sequence of the codon-optimized codingregion encoding the wild-type Thermotoga maritima acetyl xylan esterasehaving perhydrolytic activity.

SEQ ID NO: 2 is the amino acid sequence of the wild-type Thermotogamaritima acetyl xylan esterase having perhydrolytic activity.

SEQ ID NOs: 3 and 4 are the nucleic acid sequences of primers used toprepare the C277S variant acetyl xylan esterase.

SEQ ID NO: 5 is the amino acid sequence of the C277S variant acetylxylan esterase having perhydrolytic activity (Published U.S. PatentApplication No. 2010/0087529 to DiCosimo et al.).

SEQ ID NO: 6 is the nucleic acid sequence of the plasmid pSW202/C277S.

SEQ ID NOs: 7 and 8 are the nucleic acid sequences of primers used forerror-prone PCR.

SEQ ID NO: 9 is the nucleic acid sequence encoding the “A3” variantacetyl xylan esterase having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:(F24I/S35T/Q179L/N275D/C277S/S308G/F317S).

SEQ ID NO: 10 is the amino acid sequence of the “A3” variant acetylxylan esterase.

SEQ ID NOs: 11 and 12 are the nucleic acid sequences of primers used toconstruct the N275D/C277S variant acetyl xylan esterase.

SEQ ID NO: 13 is the nucleic acid sequence encoding the N275D/C277Svariant acetyl xylan esterase.

SEQ ID NO: 14 is the amino acid sequence of the N275D/C277S variantacetyl xylan esterase.

SEQ ID NOs: 15 and 16 are the nucleic acid sequences of primers used toconstruct the C277S/F317S variant acetyl xylan esterase.

SEQ ID NO: 17 is the nucleic acid sequence encoding the C277S/F317Svariant acetyl xylan esterase.

SEQ ID NO: 18 is the amino acid sequence of the C277S/F317S variantacetyl xylan esterase.

SEQ ID NOs: 19 and 20 are the nucleic acid sequences of primers used toconstruct the S35T/C277S variant acetyl xylan esterase.

SEQ ID NO: 21 is the nucleic acid sequence encoding the S35T/C277Svariant acetyl xylan esterase.

SEQ ID NO: 22 is the amino acid sequence of the S35T/C277S variantacetyl xylan esterase.

SEQ ID NOs: 23 and 24 are the nucleic acid sequences of primers used toconstruct the Q179L/C277S variant acetyl xylan esterase.

SEQ ID NO: 25 is the nucleic acid sequence encoding the Q179L/C277Svariant acetyl xylan esterase.

SEQ ID NO: 26 is the amino acid sequence of the Q179L/C277S variantacetyl xylan esterase.

DETAILED DESCRIPTION

A nucleic acid molecule encoding the Thermotoga maritima acetyl xylanesterase (SEQ ID NO: 2) was mutated by error-prone PCR and/orsite-directed mutagenesis to create a library of variant perhydrolases.Several perhydrolase variants were identified exhibiting an increase inthe ratio of peracetic acid formation (PAAF) to peracetic acidhydrolysis (PAAH) specific activities when compared to the PAAF/PAAHratio of the wild-type Thermotoga maritima perhydrolase having aminoacid sequence SEQ ID NO: 2.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an” and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

The term “comprising” means the presence of the stated features,integers, steps, or components as referred to in the claims, but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof. The term “comprising” isintended to include embodiments encompassed by the terms “consistingessentially of” and “consisting of”. Similarly, the term “consistingessentially of” is intended to include embodiments encompassed by theterm “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “multi-component system” will refer to a systemof enzymatically generating peroxycarboxylic acid wherein the componentsremain separated until use. As such, the multi-component system willinclude at least one first component that remains separated from atleast one second component. The first and second components areseparated in different compartments until use (i.e., using first andsecond compartments). The design of the multi-component systems willoften depend on the physical form of the components to be combined andare described in more detail below.

As used herein, the term “peroxycarboxylic acid” is synonymous withperacid, peroxyacid, peroxy acid, percarboxylic acid and peroxoic acid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane, 1,2,3-propanetriol triacetate and all othersynonyms of CAS Registry Number 102-76-1.

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of CASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 139-45-7.

As used herein, the term “ethyl acetate” is synonymous with aceticether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoicacid ethyl ester, ethyl acetic ester and all other synonyms of CASRegistry Number 141-78-6.

As used herein, the term “ethyl lactate” is synonymous with lactic acidethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms “acetylated sugar” and “acetylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acetylgroup. Examples include, but are not limited to, glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” mean a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In one embodiment, the hydrocarbyl moiety is astraight chain, branched or cyclic arrangement of carbon atoms connectedby single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol,1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 2,5-pentanediol,1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol, 1,6-hexanediol, referto said compounds comprising at least one ester group of the formulaRC(O)O, wherein R is a C1 to C7 linear hydrocarbyl moiety.

As used herein, the terms “suitable enzymatic reaction formulation”,“components suitable for generation of a peroxycarboxylic acid”,“suitable reaction components”, “reaction components”, “reactionformulation”, and “suitable aqueous reaction formulation” refer to thematerials and water in which the reactants and the enzyme catalystcomprising the present variant polypeptide having perhydrolytic activitycome into contact to form the desired peroxycarboxylic acid. Thecomponents of the reaction formulation are provided herein and thoseskilled in the art appreciate the range of component variations suitablefor this process. In one embodiment, the enzymatic reaction formulationproduces peroxycarboxylic acid in situ upon combining the reactioncomponents. As such, the reaction components may be provided as amulti-component system wherein one or more of the reaction componentsremains separated until use. The design of systems and means forseparating and combining multiple active components are known in the artand generally will depend upon the physical form of the individualreaction components. For example, multiple active fluids (liquid-liquid)systems typically use multi-chamber dispenser bottles or two-phasesystems (U.S. Patent Application Publication No. 200510139608; U.S. Pat.No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No. 6,391,840; E.P.Patent 0807156B1; U.S. Patent Application Publication No. 2005/0008526;and PCT Publication No. WO 00/61713A1) such as found in some bleachingapplications wherein the desired bleaching agent is produced upon mixingthe reactive fluids. Multi-component formulations and multi-componentgeneration systems to enzymatically produce peroxycarboxylic acids fromcarboxylic acid esters are described by DiCosimo et al. in Published U.SPatent Application Nos. 2010/0086510 and 2010/0086621, respectively.Other forms of multi-component systems used to generate peroxycarboxylicacid may include, but are not limited to, those designed for one or moresolid components or combinations of solid-liquid components, such aspowders used in many commercially available bleaching compositions(e.g., U.S. Pat. No. 5,116,575), multi-layered tablets (e.g., U.S. Pat.No. 6,210,639), water dissolvable packets having multiple compartments(e.g., U.S. Pat. No. 6,995,125) and solid agglomerates that react uponthe addition of water (e.g., U.S. Pat. No. 6,319,888).

As used herein, the term “substrate” or “carboxylic acid estersubstrate” will refer to the reaction components enzymaticallyperhydrolyzed using the present enzyme catalyst in the presence of asuitable source of peroxygen, such as hydrogen peroxide. In oneembodiment, the substrate comprises at least one ester group capable ofbeing enzymatically perhydrolyzed using the enzyme catalyst, whereby aperoxycarboxylic acid is produced.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with a source of hydrogen peroxide to form aperoxycarboxylic acid. Typically, inorganic peroxide is reacted with theselected substrate in the presence of a catalyst to produce theperoxycarboxylic acid. As used herein, the term “chemical perhydrolysis”includes perhydrolysis reactions in which a substrate (such as aperoxycarboxylic acid precursor) is combined with a source of hydrogenperoxide wherein peroxycarboxylic acid is formed in the absence of anenzyme catalyst. As used herein, the term “enzymatic perhydrolysis”refers a reaction of a selected substrate with a source of hydrogenperoxide to form a peroxycarboxylic acid, wherein the reaction iscatalyzed by an enzyme catalyst having perhydrolysis activity.

As used herein, the term “perhydrolase activity” refers to the enzymecatalyst activity per unit mass (for example, milligram) of protein, drycell weight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmmol of peroxycarboxylic acid product (such asperacetic acid) per minute at a specified temperature. “One unit ofenzyme activity” may also be used herein to refer to the amount ofperoxycarboxylic acid hydrolysis activity required for the hydrolysis of1 μmol of peroxycarboxylic acid (e.g., peracetic acid) per minute at aspecified temperature.

As used herein, “PAAF” means “peracetic acid formation” and refers tothe specific activity of the present enzyme catalyst for producingperacetic acid from a carboxylic acid ester substrate as measured, forexample, using triacetin.

As used herein, “PAAH” means “peracetic acid hydrolysis” and refers tothe specific activity of the present enzyme catalyst for enzymaticallyhydrolyzing peracetic acid into acetic acid and hydrogen peroxide.

As used herein, “PAAF/PAAH ratio” refers to the ratio of the specificactivities of the variant enzyme catalyst for producing peracetic acidfrom a carboxylic acid ester substrate and for hydrolyzing peraceticacid into acetic acid and hydrogen peroxide, respectively.Enzymatically-produced peracids in reaction formulations comprising anenzyme catalyst having a perhydrolytic enzyme having an increasedPAAF/PAAH ratio of specific activities are typically more stable as theperacid is less likely to be hydrolyzed to the corresponding carboxylicacid and hydrogen peroxide when the peracid formulation comprises theperhydrolytic enzyme. In one embodiment, reactions to measure peraceticacid formation (PAAF) specific activity are run at ca. 25° C. inphosphate buffer (50 mM, pH 7.2) containing 100 mM triacetin, 100 mMhydrogen peroxide and approximately 2.5 μg/mL of heat-treated extractsupernatant total protein from E. coli strain KLP18 expressing wild-typeor variant perhydrolase (see example 13). In another embodiment, thereactions to measure peracetic acid hydrolysis (PAAH) specific activityare run at ca. 25° C., phosphate buffer (50 mM, pH 7.2) containingapproximately 2000 ppm peracetic acid (ca. 26.3 mM) and 25 μg/mL ofheat-treated extract supernatant total protein from E. coli strain KLP18expressing wild-type or variant perhydrolase (see Example 14).

As used herein, the “fold increase” in PAAF/PAAH ratio of specificactivities is measured relative to the PAAF/PAAH ratio of the Thermotogamaritima wild-type perhydrolase (SEQ ID NO: 2) under the same reactionconditions. In one embodiment, the fold increase in the PAAF/PAAH ratioof the variant polypeptide (i.e., variant perhydrolase) relative to theThermotoga maritima wild-type perhydrolase is at least 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9., 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,or 10-fold when compared under identical reaction/assay conditions. Inanother embodiment, the fold increase in the PAAF/PAAH ratio of thevariant enzyme may be measured relative to the C277S variantperhydrolase of SEQ ID NO: 5 (U.S. patent application Ser. No.12/572,094) and is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9., 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold when comparedunder identical reaction conditions.

As used herein, “identical assay conditions” or “same assay conditions”refer to the conditions used to measure the peracid formation (i.e.,perhydrolysis of a carboxylic acid ester substrate) specific activity orthe peroxycarboxylic acid hydrolysis specific activity of the variantpolypeptide relative to the respective specific activities of thepolypeptide from which is was derived (i.e., Thermotoga maritimawild-type acetyl xylan esterase of SEQ ID NO: 2). The assay conditionsused to measure the respective specific activities should be as close toidentical as possible such that only the structure of the polypeptidehaving perhydrolytic activity varies. The carboxylic acid estersubstrate and the corresponding peroxycarboxylic acid used to measurethe respective perhydrolytic specific activity and peracid hydrolysisspecific activities may vary depending upon the desiredsubstrate/product combination. In one embodiment, the perhydrolyticspecific activity is measured using triacetin as a substrate and theperacid hydrolysis specific activity is measured using peracetic acid asthe respective peracid. In one embodiment, reactions used to measureperacetic acid formation (PAAF) specific activity are run at ca. 25° C.in phosphate buffer (50 mM, pH 7.2) containing 100 mM triacetin, 100 mMhydrogen peroxide and approximately 2.5 μg/mL of heat-treated extractsupernatant total protein from E. coli strain KLP18 expressing wild-typeor variant perhydrolase (see example 13). In another embodiment, thereactions to measure peracetic acid hydrolysis (PAAH) specific activityare run at ca. 25° C., phosphate buffer (50 mM, pH 7.2) containingapproximately 2000 ppm peracetic acid (ca. 26.3 mM) and 25 μg/mL ofheat-treated extract supernatant total protein from E. coli strain KLP18expressing wild-type or variant perhydrolase (see Example 14).

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme (i.e., a polypeptide) havingperhydrolysis activity and may be in the form of a whole microbial cell,permeabilized microbial cell(s), one or more cell components of amicrobial cell extract, partially purified enzyme, or purified enzyme.The enzyme catalyst may also be chemically modified (for example, bypegylation or by reaction with cross-linking reagents). The perhydrolasecatalyst may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997.

The present enzyme catalyst comprises a variant polypeptide havingperhydrolytic activity and is structurally classified as a member of thecarbohydrate family esterase family 7 (CE-7 family) of enzymes (seeCoutinho, P. M., Henrissat, B. “Carbohydrate-active enzymes: anintegrated database approach” in Recent Advances in CarbohydrateBioengineering, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.). TheCE-7 family of enzymes has been demonstrated to be particularlyeffective for producing peroxycarboxylic acids from a variety ofcarboxylic acid ester substrates when combined with a source ofperoxygen (See PCT publication No. WO2007/070609 and U.S. PatentApplication Publication Nos. 2008/0176299, 2008/176783, and 2009/0005590to DiCosimo et al.; each herein incorporated by reference in theirentireties). The CE-7 enzyme family includes cephalosporin Cdeacetylases (CAHs; E.C. 3.1.1.41) and acetyl xylan esterases (AXEs;E.C. 3.1.1.72). Members of the CE-7 enzyme family share a conservedsignature motif (Vincent et al., J. Mol. Biol., 330:593-606 (2003)).

As used herein, the terms “signature motif” and “CE-7 signature motif”,refer to conserved structures shared among a family of enzymes having aperhydrolytic activity.

As used herein, “structurally, classified as a CE-7 enzyme”,“structurally classified as a carbohydrate esterase family 7 enzyme”,“structurally classified as a CE-7 carbohydrate esterase”, and “CE-7perhydrolase” will be used to refer to enzymes having perhydrolysisactivity that are structurally classified as a CE-7 carbohydrateesterase based on the presence of the CE-7 signature motif (Vincent etal., supra). The “signature motif” for CE-7 esterases comprises threeconserved motifs (residue position numbering relative to referencesequence SEQ ID NO: 2; the wild-type Thermotoga maritima acetyl xylanesterase):

a) Arg118-Gly119-Gln120;

b) Gly186-Xaa187-Ser188-Gln189-Gly190; and

c) His303-Glu304.

Typically, the Xaa at amino acid residue position 187 is glycine,alanine, proline, tryptophan, or threonine. Two of the three amino acidresidues belonging to the catalytic triad are in bold. In oneembodiment, the Xaa at amino acid residue position 187 is selected fromthe group consisting of glycine, alanine, proline, tryptophan, andthreonine.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 272-274 of SEQ ID NO: 2) that may be usedto further define a member of the CE-7 carbohydrate esterase family. Ina further embodiment, the signature motif defined above includes afourth conserved motif defined as:

Leu272-Xaa273-Asp274.

The Xaa at amino acid residue position 273 is typically isoleucine,valine, or methionine. The fourth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser188-Asp274-His303).

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., Appl. Environ.Microbiol., 61(6): 2224-2229 (1995); U.S. Pat. No. 5,528,152; and U.S.Pat. No. 5,338,676). Enzymes classified as cephalosporin C deacetylaseshave been shown to often have significant perhydrolytic activity (U.S.Patent Application Publication Nos. 2008-0176783 and 2008-0176299 toDiCosimo et al.).

As used herein, “acetyl xylan esterase” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides. Enzymes classified as acetyl xylanesterases have been shown to have significant perhydrolytic activity(U.S. Patent Application Publication Nos. 2008-0176783, 2008-0176299,and 2009/0005590, each to DiCosimo et al.).

As used herein, the term “Thermotoga maritima” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®NP_(—)227893.1). In one aspect, the Thermotoga maritima strain isThermotoga maritima MSB8. The amino acid sequence of the wild-typeenzyme having perhydrolase activity from Thermotoga maritima is providedas SEQ ID NO: 2.

As used herein, the terms “variant”, “variant polypeptide”, and “variantenzyme catalyst” refer to an enzyme catalyst comprising at least onepolypeptide (i.e., a perhydrolase) having perhydrolytic activity whereinthe polypeptide comprises at least one amino acid change relative to theenzyme/polypeptide from which it was derived (typically the wild-typeperhydrolase). Several variant polypeptides are provided herein havingperhydrolytic activity and are characterized by an increase in thePAAF/PAAH ratio relative to the Thermotoga maritima wild-type acetylxylan esterase having amino acid sequence SEQ ID NO: 2.

For a particular variant perhydrolase, amino acid substitutions arespecified with reference to the Thermotoga maritima amino acid sequence(SEQ ID NO: 2). The wild-type amino acid (denoted by the standard singleletter abbreviation) is followed by the amino acid residue position ofSEQ ID NO: 2 followed by the amino acid of the variant (also denoted bythe standard single letter abbreviation). For example, “C277S” describesa change in SEQ ID NO: 2 at amino acid residue position 277 wherecysteine was changed to serine. The variant polypeptide may be comprisedof multiple point substitutions. For example, N275D/C277S refers to avariant polypeptide having two point substitutions: 1) a change at aminoacid residue position 275 where an asparagine was changed to asparticacid, and 2) a change at residue position 277 wherein a cysteine waschanged to a serine.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid (or asdefined herein) Xaa X

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to microorganisms, spores, viruses, prions, and mixturesthereof. The present enzyme can be used to produce an efficaciousconcentration of at least one peroxycarboxylic acid useful to reduceand/or eliminate the presence of the viable biological contaminants. Ina preferred embodiment, the biological contaminant is a viablepathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces. As used herein, the term “antiseptic”refers to a chemical agent that inhibits the growth of disease-carryingmicroorganisms. In one aspect of the embodiment, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “sanitary” means of or relating to therestoration or preservation of health, typically by removing, preventingor controlling an agent that may be injurious to health. As used herein,the term “sanitize” means to make sanitary. As used herein, the term“sanitizer” refers to a sanitizing agent. As used herein the term“sanitization” refers to the act or process of sanitizing.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peroxycarboxylic acids can have virucidalactivity. Typical alternative virucides known in the art which may besuitable for use with the present invention include, for example,alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactone,iodine, chlorine, mercury salts, hydroxylamine, ethylene oxide, ethyleneglycol, quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peroxycarboxylic acids canhave biocidal activity. Typical alternative biocides known in the art,which may be suitable for use in the present invention include, forexample, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites,ozone, acrolein, amines, chlorinated phenolics, copper salts,organo-sulphur compounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3-log reduction, morepreferably at least a 4-log reduction, and most preferably at least a5-log reduction. In another aspect, the minimum biocidal concentrationis at least a 6-log reduction in viable microbial cells.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to, hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates. As described herein, the concentration ofthe hydrogen peroxide provided by the peroxygen compound in the aqueousreaction formulation is initially at least 1 mM or more upon combiningthe reaction components. In one embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is at least 10 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction formulation is at least 100 mM. In another embodiment, thehydrogen peroxide concentration in the aqueous reaction formulation isat least 200 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is 500 mM or more. Inyet another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is 1000 mM or more. The molar ratio of thehydrogen peroxide to enzyme substrate, such as triglyceride,(H₂O₂:substrate) in the aqueous reaction formulation may be from about0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5to 5.

As used herein, the term “benefit agent” refers to a material thatpromotes or enhances a useful advantage, a favorable/desirable effect orbenefit. In one embodiment, a process is provided whereby a benefitagent, such as a composition comprising a peroxycarboxylic acid, isapplied to a textile or article of clothing to achieve a desiredbenefit, such as disinfecting, bleaching, destaining, deodorizing, andany combination thereof.

Variant Polypeptides Having an Increased PAAF/PAAH Ratio.

The present variant polypeptides were derived from the Thermotogamaritima wild-type acetyl xylan esterase that has been previouslydemonstrated to have significant perhydrolytic activity for producingperoxycarboxylic acids from carboxylic acid esters and a source ofperoxygen, such as hydrogen peroxide (U.S. Patent ApplicationPublication No. 2008-0176299 to DiCosimo et al.). However, theThermotoga maritima wild-type acetyl xylan esterase also has the abilityto hydrolyze the peroxycarboxylic acid product into the correspondingcarboxylic acid and hydrogen peroxide.

Given that the desired peroxycarboxylic acid-based bleaching ordisinfecting formulation comprising enzymatically-producedperoxycarboxylic acid will likely contain at least some of the activeenzyme catalyst, there is a need to identify at least one variantpolypeptide having a higher PAAF/PAAH ratio when compared to thePAAF/PAAH ratio of the Thermotoga maritima wild-type enzyme under thesame (or as reasonably identical as possible) assay conditions. In oneembodiment, the increase in the PAAF/PAAH ratio preferably occurs acrossthe pH range where the enzyme is typically active. In anotherembodiment, the increase in the PAAF/PAAH ratio preferably occurswithout a substantial drop in the perhydrolysis to hydrolysis reaction(P/H) ratio of the enzyme for the carboxylic acid ester substrate (i.e.,the perhydrolytic reactions typically occur in an aqueous reactionformulation where the carboxylic acid ester may be hydrolyzed by theenzyme catalyst).

A library of variant polypeptides was created from the wild-typeThermotoga maritima perhydrolase (SEQ ID NO: 2) and assayed for anincrease in the ratio of perhydrolytic specific activity relative to theperoxycarboxylic acid hydrolysis activity. In order to measure thisratio, an assay was developed to measure the specific activity of thevariant polypeptide for peracetic acid formation (“PAAF”) from triacetin(100 mM) and hydrogen peroxide (100 mM) at ca. 25° C. in phosphatebuffer (50 mM, pH 7.2) using heat-treated extract supernatant totalprotein produced in E. coli strain KLP18 expressing wild-type or variantperhydrolase (Example 13). The specific activity of the variantpolypeptide for hydrolyzing the peroxycarboxylic acid (PAAH) wasmeasured using ca. 2000 ppm peracetic acid (ca. 26.3 mM) in phosphatebuffer (50 mM, pH 7.2) at ca. 25° C. using heat-treated extractsupernatant total protein from E. coli strain KLP18 expressing wild-typeor variant perhydrolase (see Example 14). The PAAF specific activity wasdivided by the PAAH specific activity to determine the PAAF/PAAH ratio.

Suitable Reaction Conditions for the Enzyme-Catalyzed Preparation ofPeroxycarboxylic Acids from Carboxylic Acid Esters and Hydrogen Peroxide

A process is provided to produce an aqueous formulation comprising atleast one peroxycarboxylic acid by reacting carboxylic acid esters andan inorganic peroxide (such as hydrogen peroxide, sodium perborate orsodium percarbonate) in the presence of an enzyme catalyst havingperhydrolysis activity, wherein the enzyme catalyst comprises an enzymehaving amino acid sequence SEQ ID NO: 26. Although the increase in thePAAF/PAAH ratio was determined using a controlled set of specificreaction conditions, the variant enzyme catalyst may be used to produceperoxycarboxylic acids from any number of suitable substrates under avariety of reaction conditions.

In one embodiment, suitable substrates include one or more estersprovided by the following formula:

[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O

R₆═C1 to C7 linear, branched or cyclic hydrocarbyl moiety, optionallysubstituted with hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆optionally comprises one or more ether linkages for R₆=C2 to C7;

R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety optionallysubstituted with hydroxyl groups; wherein each carbon atom in R₅individually comprises no more than one hydroxyl group or no more thanone ester group; wherein R₅ optionally comprises one or more etherlinkages;

m=1 to the number of carbon atoms in R₅; and

-   -   wherein said esters have solubility in water of at least 5 ppm        at 25° C.

In another embodiment, R₆ is C1 to C7 linear hydrocarbyl moiety,optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups,optionally comprising one or more ether linkages. In a further preferredembodiment, R₆ is C2 to C7 linear hydrocarbyl moiety, optionallysubstituted with hydroxyl groups, and/or optionally comprising one ormore ether linkages.

In another embodiment, suitable substrates also include one or moreglycerides of the formula:

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O).

In another aspect, suitable substrates may also include one or moreesters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10.

Suitable substrates may also include one or more acetylated saccharidesselected from the group consisting of acetylated mono-, di-, andpolysaccharides. In another embodiment, the acetylated saccharides areselected from the group consisting of acetylated xylan, fragments ofacetylated xylan, acetylated xylose (such as xylose tetraacetate),acetylated glucose (such as glucose pentaacetate),β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-D-glucal, and acetylated cellulose. In a preferredembodiment, the acetylated saccharide is selected from the groupconsisting of β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetylatedcellulose.

In another embodiment, suitable substrates are selected from the groupconsisting of: monoacetin; diacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylanfragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; and mixtures thereof.

In another embodiment, the carboxylic acid ester is selected from thegroup consisting of monoacetin, diacetin, triacetin, and combinationsthereof. In another embodiment, the substrate is a C1 to C6 polyolcomprising one or more ester groups. In a preferred embodiment, one ormore of the hydroxyl groups on the C1 to C6 polyol are substituted withone or more acetoxy groups (such as 1,3-propanediol diacetate,1,4-butanediol diacetate, etc.). In a further embodiment, the substrateis propylene glycol diacetate (PGDA), ethylene glycol diacetate (EGDA),or a mixture thereof.

In another embodiment, suitable substrates are selected from the groupconsisting of ethyl acetate; methyl lactate; ethyl lactate; methylglycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate;methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; triethyl 2-acetylcitrate; glucose pentaacetate; gluconolactone; glycerides (mono-, di-,and triglycerides) such as monoacetin, diacetin, triacetin,monopropionin, dipropionin (glyceryl dipropionate), tripropionin(1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryldibutyrate), tributyrin (1,2,3-tributyrylglycerol); acetylatedsaccharides; and mixtures thereof.

In a further embodiment, suitable substrates are selected from the groupconsisting of monoacetin, diacetin, triacetin, monopropionin,dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, ethylacetate, and ethyl lactate. In yet another aspect, the substrate isselected from the group consisting of diacetin, triacetin, ethylacetate, and ethyl lactate. In a most preferred embodiment, the suitablesubstrate comprises triacetin.

The carboxylic acid ester is present in the aqueous reaction formulationat a concentration sufficient to produce the desired concentration ofperoxycarboxylic acid upon enzyme-catalyzed perhydrolysis. Thecarboxylic acid ester need not be completely soluble in the aqueousreaction formulation, but has sufficient solubility to permit conversionof the ester by the perhydrolase catalyst to the correspondingperoxycarboxylic acid. The carboxylic acid ester is present in theaqueous reaction formulation at a concentration of 0.0005 wt % to 40 wt% of the aqueous reaction formulation, preferably at a concentration of0.01 wt % to 20 wt % of the aqueous reaction formulation, and morepreferably at a concentration of 0.05 wt % to 10 wt % of the aqueousreaction formulation. The wt % of carboxylic acid ester may optionallybe greater than the solubility limit of the carboxylic acid ester, suchthat the concentration of the carboxylic acid ester is at least 0.0005wt % in the aqueous reaction formulation that is comprised of water,enzyme catalyst, and source of peroxide, where the remainder of thecarboxylic acid ester remains as a second separate phase of a two-phaseaqueous/organic reaction formulation. Not all of the added carboxylicacid ester must immediately dissolve in the aqueous reactionformulation, and after an initial mixing of all reaction components,additional continuous or discontinuous mixing is optional.

The peroxycarboxylic acids produced by the present reaction componentsmay vary depending upon the selected substrates, so long as the presentenzyme catalyst is used. In one embodiment, the peroxycarboxylic acidproduced is peracetic acid, perpropionic acid, perbutyric acid,perlactic acid, perglycolic acid, permethoxyacetic acid,per-β-hydroxybutyric acid, or mixtures thereof.

The peroxygen source may include, but is not limited to, hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)), perborate salts and percarbonate salts. Theconcentration of peroxygen compound in the aqueous reaction formulationmay range from 0.0033 wt % to about 50 wt %, preferably from 0.033 wt %to about 40 wt %, more preferably from 0.33 wt to about 30 wt %.

Many perhydrolase catalysts (such as whole cells, permeabilized wholecells, and partially purified whole cell extracts) have been reported tohave catalase activity (EC 1.11.1.6). Catalases catalyze the conversionof hydrogen peroxide into oxygen and water. In one aspect, the enzymecatalyst having perhydrolase activity lacks catalase activity. Inanother aspect, a catalase inhibitor is added to the aqueous reactionformulation. Examples of catalase inhibitors include, but are notlimited to, sodium azide and hydroxylamine sulfate. One of skill in theart can adjust the concentration of catalase inhibitor as needed. Theconcentration of the catalase inhibitor typically ranges from 0.1 mM toabout 1 M; preferably about 1 mM to about 50 mM; more preferably fromabout 1 mM to about 20 mM. In one aspect, sodium azide concentrationtypically ranges from about 20 mM to about 60 mM while hydroxylaminesulfate is concentration is typically about 0.5 mM to about 30 mM,preferably about 10 mM.

The catalase activity in a host cell can be down-regulated or eliminatedby disrupting expression of the gene(s) responsible for the catalaseactivity using well known techniques including, but not limited to,transposon mutagenesis, RNA antisense expression, targeted mutagenesis,and random mutagenesis. In a preferred embodiment, the gene(s) encodingthe endogenous catalase activity are down-regulated or disrupted (i.e.,“knocked-out”). As used herein, a “disrupted” gene is one where theactivity and/or function of the protein encoded by the modified gene isno longer present. Means to disrupt a gene are well-known in the art andmay include, but are not limited to, insertions, deletions, or mutationsto the gene so long as the activity and/or function of the correspondingprotein is no longer present. In a further preferred embodiment, theproduction host is an E. coli production host comprising a disruptedcatalase gene selected from the group consisting of katG and katE (seeU.S. Patent Application Publication No. 2008-0176783 to DiCosimo etal.). In another embodiment, the production host is an E. coli straincomprising a down-regulation and/or disruption in both katG and katEcatalase genes. An E. coli strain comprising a double-knockout of katGand katE has been prepared and is described as E. coli strain KLP18(U.S. Patent Application Publication No. 2008-0176783 to DiCosimo atal.).

The concentration of the catalyst in the aqueous reaction formulationdepends on the specific catalytic activity of the catalyst, and ischosen to obtain the desired rate of reaction. The weight of catalyst inperhydrolysis reactions typically ranges from 0.0001 mg to 50 mg per mLof total reaction volume, preferably from 0.0005 mg to 10. mg per mL,more preferably from 0.0010 mg to 2.0 mg per mL The catalyst may also beimmobilized on a soluble or insoluble support using methods well-knownto those skilled in the art; see for example, Immobilization of Enzymesand Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J.,USA; 1997. The use of immobilized catalysts permits the recovery andreuse of the catalyst in subsequent reactions. The enzyme catalyst maybe in the form of whole microbial cells, permeabilized microbial cells,microbial cell extracts, partially-purified or purified enzymes, andmixtures thereof.

In one aspect, the concentration of peroxycarboxylic acid generated bythe combination of chemical perhydrolysis and enzymatic perhydrolysis ofthe carboxylic acid ester is sufficient to provide an effectiveconcentration of peroxycarboxylic acid for disinfection, bleaching,sanitization, deodorizing or destaining at a desired pH. In anotheraspect, the present methods provide combinations of enzymes and enzymesubstrates to produce the desired effective concentration ofperoxycarboxylic acid, where, in the absence of added enzyme, there is asignificantly lower concentration of peroxycarboxylic acid produced.Although there may be some chemical perhydrolysis of the enzymesubstrate by direct chemical reaction of inorganic peroxide with theenzyme substrate, there may not be a sufficient concentration ofperoxycarboxylic acid generated to provide an effective concentration ofperoxycarboxylic acid in the desired applications, and a significantincrease in total peroxycarboxylic acid concentration is achieved by theaddition of an appropriate perhydrolase catalyst to the aqueous reactionformulation.

In one aspect of the invention, the concentration of peroxycarboxylicacid generated (e.g. peracetic acid) by the enzymatic perhydrolysis isat least about 2 ppm, preferably at least 20 ppm, preferably at least100 ppm, more preferably at least about 200 ppm peroxycarboxylic acid,more preferably at least 300 ppm, more preferably at least 500 ppm, morepreferably at least 700 ppm, more preferably at least about 1000 ppmperoxycarboxylic acid, most preferably at least 2000 ppmperoxycarboxylic acid within 5 minutes more preferably within 1 minuteof initiating the enzymatic perhydrolysis reaction. In a second aspectof the invention, the concentration of peroxycarboxylic acid generated(e.g. peracetic acid) by the enzymatic perhydrolysis is at least about 2ppm, preferably at least 20 ppm, preferably at least 30 ppm, morepreferably at least about 40 ppm peroxycarboxylic acid, more preferablyat least 50 ppm, more preferably at least 60 ppm, more preferably atleast 70 ppm, more preferably at least about 80 ppm peroxycarboxylicacid, most preferably at least 100 ppm peroxycarboxylic acid within 5minutes, more preferably within 1 minute, of initiating the enzymaticperhydrolysis reaction (i.e., time measured from combining the reactioncomponents to form the formulation).

The aqueous formulation comprising the peroxycarboxylic acid may beoptionally diluted with diluent comprising water, or a solutionpredominantly comprised of water, to produce a formulation with thedesired lower target concentration of peroxycarboxylic acid. In oneaspect, the reaction time required to produce the desired concentration(or concentration range) of peroxycarboxylic acid is about 5 minutes orless, more preferably about 1 minute or less.

In other aspects, the surface or inanimate object contaminated with aconcentration of a biological contaminant(s) is contacted with theperoxycarboxylic acid formed in accordance with the processes describedherein within about 1 minute to about 168 hours of combining saidreaction components, or within about 1 minute to about 48 hours, orwithin about 1 minute to 2 hours of combining said reaction components,or any such time interval therein.

In another aspect, the peroxycarboxylic acid formed in accordance withthe processes describe herein is used in a laundry care applicationwherein the peroxycarboxylic acid is contacted with clothing or atextile to provide a benefit, such as disinfecting, bleaching,destaining, deodorizing and/or a combination thereof. Theperoxycarboxylic acid may be used in a variety of laundry care productsincluding, but not limited to, laundry or textile pre-wash treatments,laundry detergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents. In one embodiment, thepresent process to produce a peroxycarboxylic acid for a target surfaceis conducted in situ.

In the context of laundry care applications, the term “contacting anarticle of clothing or textile” means that the article of clothing ortextile is exposed to a formulation disclosed herein. To this end, thereare a number of formats the formulation may be used to treat articles ofclothing or textiles including, but not limited to, liquid, solids, gel,paste, bars, tablets, spray, foam, powder, or granules and can bedelivered via hand dosing, unit dosing, dosing from a substrate,spraying and automatic dosing from a laundry washing or drying machine.Granular compositions can also be in compact form; liquid compositionscan also be in a concentrated form.

When the formulations disclosed herein are used in a laundry washingmachine, the formulation can further contain components typical tolaundry detergents. For example, typical components included, but arenot limited to, surfactants, bleaching agents, bleach activators,additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents, softeningagents, corrosion inhibitors, tarnish inhibitors, germicides, pHadjusting agents, non-builder alkalinity sources, chelating agents,organic and/or inorganic fillers, solvents, hydrotropes, opticalbrighteners, dyes, and perfumes.

The formulations disclosed herein can also be used as detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

In connection with the present systems and methods for laundry carewhere the peracid is generated for one or more of bleaching, stainremoval, and odor reduction, the concentration of peracid generated(e.g., peracetic acid) by the perhydrolysis of at least one carboxylicacid ester may be at least about 2 ppm, preferably at least 20 ppm,preferably at least 100 ppm, and more preferably at least about 200 ppmperacid. In connection with the present systems and methods for laundrycare where the peracid is generated for disinfection or sanitization,the concentration of peracid generated (e.g., peracetic acid) by theperhydrolysis of at least one carboxylic acid ester may be at leastabout 2 ppm, more preferably at least 20 ppm, more preferably at least200 ppm, more preferably at least 500 ppm, more preferably at least 700ppm, more preferably at least about 1000 ppm peracid, most preferably atleast 2000 ppm peracid within 10 minutes, preferably within 5 minutes,and most preferably within 1 minute of initiating the perhydrolysisreaction. The product formulation comprising the peracid may beoptionally diluted with water, or a solution predominantly comprised ofwater, to produce a formulation with the desired lower concentration ofperacid. In one aspect of the present methods and systems, the reactiontime required to produce the desired concentration of peracid is notgreater than about two hours, preferably not greater than about 30minutes, more preferably not greater than about 10 minutes, even morepreferably not greater than about 5 minutes, and most preferably inabout 1 minute or less.

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of theaqueous reaction formulation (approximately 0° C.) to about 85° C., witha preferred range of reaction temperature of from about 5° C. to about55° C.

The pH of the aqueous reaction formulation while enzymatically producingperoxycarboxylic acid is maintained at a pH ranging from about 5.0 toabout 10.0, preferably about 6.5 to about 8.5, and yet even morepreferably about 6.5 to about 7.5. In one embodiment, the pH of theaqueous reaction formulation ranges from about 6.5 to about 8.5 for atleast 30 minutes after combining the reaction components. The pH of theaqueous reaction formulation may be adjusted or controlled by theaddition or incorporation of a suitable buffer, including, but notlimited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate.In one embodiment, the buffer is selected from a phosphate buffer and abicarbonate buffer. The concentration of buffer, when employed, istypically from 0.1 mM to 1.0 M, preferably from 1 mM to 300 mM, mostpreferably from 10 mM to 100 mM. In another aspect of the presentinvention, no buffer is added to the reaction mixture whileenzymatically producing peroxycarboxylic acid.

In yet another aspect, the enzymatic perhydrolysis aqueous reactionformulation may contain an organic solvent that acts as a dispersant toenhance the rate of dissolution of the carboxylic acid ester in theaqueous reaction formulation. Such solvents include, but are not limitedto, propylene glycol methyl ether, acetone, cyclohexanone, diethyleneglycol butyl ether, tripropylene glycol methyl ether, diethylene glycolmethyl ether, propylene glycol butyl ether, dipropylene glycol methylether, cyclohexanol, benzyl alcohol, isopropanol, ethanol, propyleneglycol, and mixtures thereof.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to, buffers,detergent builders, thickening agents, emulsifiers, surfactants, wettingagents, corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers,and peroxide stabilizers (e.g., metal ion chelating agents). Many of theadditional components are well known in the detergent industry (see, forexample, U.S. Pat. No. 5,932,532; hereby incorporated by reference).Examples of emulsifiers include, but are not limited to, polyvinylalcohol or polyvinylpyrrolidone. Examples of thickening agents include,but are not limited to, LAPONITE® RD, corn starch, PVP, CARBOWAX®,CARBOPOL®, CABOSIL®, polysorbate 20, PVA, and lecithin. Examples ofbuffering systems include, but are not limited to, sodium phosphatemonobasic/sodium phosphate dibasic; sulfamic acid/triethanolamine;citric acid/triethanolamine; tartaric acid/triethanolamine; succinicacid/triethanolamine; and acetic acid/triethanolamine. Examples ofsurfactants include, but are not limited to, a) non-ionic surfactantssuch as block copolymers of ethylene oxide or propylene oxide,ethoxylated or propoxylated linear and branched primary and secondaryalcohols, and aliphatic phosphine oxides b) cationic surfactants such asquaternary ammonium compounds, particularly quaternary ammoniumcompounds having a C8-C20 alkyl group bound to a nitrogen atomadditionally bound to three C1-C2 alkyl groups, c) anionic surfactantssuch as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkylphosphonates, alkane sulfonates (e.g., sodium dodecylsulphate “SDS”) orlinear or branched alkyl benzene sulfonates, alkene sulfonates and d)amphoteric and zwitterionic surfactants such as aminocarboxylic acids,aminodicarboxylic acids, alkybetaines, and mixtures thereof. Additionalcomponents may include fragrances, dyes, stabilizers of hydrogenperoxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST®2010, Solutia Inc.,St. Louis, Mo.) and ethylenediaminetetraacetic acid (EDTA)), TURPINAL®SL, DEQUEST® 0520, DEQUEST® 0531, stabilizers of enzyme activity (e.g.,polyethylene glycol (PEG)), and detergent builders.

In another aspect, the enzymatic perhydrolysis product may be pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected.

In another aspect, the enzymatic perhydrolysis product is not pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected, butinstead, the components of the aqueous reaction formulation thatgenerate the desired concentration of peroxycarboxylic acid arecontacted with the surface or inanimate object to be disinfected and/orbleached or destained, generating the desired concentration ofperoxycarboxylic acid. In some embodiments, the components of theaqueous reaction formulation combine or mix at the locus. In someembodiments, the reaction components are delivered or applied to thelocus and subsequently mix or combine to generate the desiredconcentration of peroxycarboxylic acid.

Production of Peroxycarboxylic Acids Using a Perhydrolase Catalyst

The peroxycarboxylic acids, once produced, are quite reactive and maydecrease in concentration over extended periods of time, depending onvariables that include, but are not limited to, temperature and pH. Assuch, it may be desirable to keep the various reaction componentsseparated, especially for liquid formulations. In one aspect, thehydrogen peroxide source is separate from either the substrate or theperhydrolase catalyst, preferably from both. This can be accomplishedusing a variety of techniques including, but not limited to, the use ofmulticompartment chambered dispensers (U.S. Pat. No. 4,585,150) and atthe time of use physically combining the perhydrolase catalyst with asource of peroxygen (such as hydrogen peroxide) and the presentsubstrates to initiate the aqueous enzymatic perhydrolysis reaction. Theperhydrolase catalyst may optionally be immobilized within the body ofreaction chamber or separated (e.g., filtered, etc.) from the reactionproduct comprising the peroxycarboxylic acid prior to contacting thesurface and/or object targeted for treatment. The perhydrolase catalystmay be in a liquid matrix or in a solid form (e.g., powder or tablet) orembedded within a solid matrix that is subsequently mixed with thesubstrates to initiate the enzymatic perhydrolysis reaction. In afurther aspect, the perhydrolase catalyst may be contained within adissolvable or porous pouch that may be added to the aqueous substratematrix to initiate enzymatic perhydrolysis. In yet a further aspect, theperhydrolase catalyst may comprise the contents contained within aseparate compartment of a dissolvable or porous pouch that has at leastone additional compartment for the containment contents comprising theester substrate and/or source of peroxide. In an additional furtheraspect, a powder comprising the enzyme catalyst is suspended in thesubstrate (e.g., triacetin), and at time of use is mixed with a sourceof peroxygen in water.

Method for Determining the Concentration of Peroxycarboxylic Acid andHydrogen Peroxide.

A variety of analytical methods can be used in the present method toanalyze the reactants and products including, but not limited to,titration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the analytical procedure described by U. Karst et al. (Anal.Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S.Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO2004/058961 A1) as described in U.S. Patent Application Publication No.2008/0176783.

Determination of Minimum Biocidal Concentration of PeroxycarboxylicAcids

The method described by J. Gabrielson et al. (J. Microbiol. Methods 50:63-73 (2002)) can be employed for determination of the Minimum BiocidalConcentration (MBC) of peroxycarboxylic acids, or of hydrogen peroxideand enzyme substrates. The assay method is based on XTT reductioninhibition, where XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to, viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically Prepared Peroxycarboxylic Acid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced accordingto the present method can be used in a variety of hard surface/inanimateobject applications for reduction of concentrations of biologicalcontaminants, such as decontamination of medical instruments (e.g.,endoscopes), textiles (such as garments and carpets), food preparationsurfaces, food storage and food-packaging equipment, materials used forthe packaging of food products, chicken hatcheries and grow-outfacilities, animal enclosures, and spent process waters that havemicrobial and/or virucidal activity. The enzyme-generatedperoxycarboxylic acids may be used in formulations designed toinactivate prions (e.g., certain proteases) to additionally providebiocidal activity (see U.S. Pat. No. 7,550,420 to DiCosimo et al.).

In one aspect, the peroxycarboxylic acid composition is useful as adisinfecting agent for non-autoclavable medical instruments and foodpackaging equipment. As the peroxycarboxylic acid-containing formulationmay be prepared using GRAS or food-grade components (enzyme, enzymesubstrate, hydrogen peroxide, and buffer), the enzyme-generatedperoxycarboxylic acid may also be used for decontamination of animalcarcasses, meat, fruits and vegetables, or for decontamination ofprepared foods. The enzyme-generated peroxycarboxylic acid may beincorporated into a product whose final form is a powder, liquid, gel,film, solid or aerosol. The enzyme-generated peroxycarboxylic acid maybe diluted to a concentration that still provides an efficaciousdecontamination.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can be used to disinfect surfaces and/or objectscontaminated (or suspected of being contaminated) with biologicalcontaminants, such as pathogenic microbial contaminants, by contactingthe surface or object with the products produced by the presentprocesses. As used herein, “contacting” refers to placing a disinfectingcomposition comprising an effective concentration of peroxycarboxylicacid in contact with the surface or inanimate object suspected ofcontamination with a biological contaminant for a period of timesufficient to clean and disinfect. Contacting includes spraying,treating, immersing, flushing, pouring on or in, mixing, combining,painting, coating, applying, affixing to and otherwise communicating aperoxycarboxylic acid solution or composition comprising an efficaciousconcentration of peroxycarboxylic acid, or a solution or compositionthat forms an efficacious concentration of peroxycarboxylic acid, withthe surface or inanimate object suspected of being contaminated with aconcentration of a biological contaminant. The disinfectant compositionsmay be combined with a cleaning composition to provide both cleaning anddisinfection. Alternatively, a cleaning agent (e.g., a surfactant ordetergent) may be incorporated into the formulation to provide bothcleaning and disinfection in a single composition.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can also contain at least one additionalantimicrobial agent, combinations of prion-degrading proteases, avirucide, a sporicide, or a biocide. Combinations of these agents withthe peroxycarboxylic acid produced by the claimed processes can providefor increased and/or synergistic effects when used to clean anddisinfect surfaces and/or objects contaminated (or suspected of beingcontaminated) with biological contaminants. Suitable antimicrobialagents include carboxylic esters (e.g., p-hydroxy alkyl benzoates andalkyl cinnamates), sulfonic acids (e.g., dodecylbenzene sulfonic acid),iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite), organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof, phenolic derivatives (e.g., o-phenylphenol, o-benzyl-p-chlorophenol, tert-amyl phenol and C₁-C₆ alkylhydroxy benzoates), quaternary ammonium compounds (e.g.,alkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof), and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peroxycarboxylic acids formed by the process can beused to reduce the concentration of viable biological contaminants (suchas a microbial population) when applied on and/or at a locus. As usedherein, a “locus” comprises part or all of a target surface suitable fordisinfecting or bleaching. Target surfaces include all surfaces that canpotentially be contaminated with biological contaminants. Non-limitingexamples include equipment surfaces found in the food or beverageindustry (such as tanks, conveyors, floors, drains, coolers, freezers,equipment surfaces, walls, valves, belts, pipes, drains, joints,crevasses, combinations thereof, and the like); building surfaces (suchas walls, floors and windows); non-food-industry related pipes anddrains, including water treatment facilities, pools and spas, andfermentation tanks; hospital or veterinary surfaces (such as walls,floors, beds, equipment (such as endoscopes), clothing worn inhospital/veterinary or other healthcare settings, including clothing,scrubs, shoes, and other hospital or veterinary surfaces); restaurantsurfaces; bathroom surfaces; toilets; clothes and shoes; surfaces ofbarns or stables for livestock, such as poultry, cattle, dairy cows,goats, horses and pigs; hatcheries for poultry or for shrimp; andpharmaceutical or biopharmaceutical surfaces (e.g., pharmaceutical orbiopharmaceutical manufacturing equipment, pharmaceutical orbiopharmaceutical ingredients, pharmaceutical or biopharmaceuticalexcipients). Additional hard surfaces include food products, such asbeef, poultry, pork, vegetables, fruits, seafood, combinations thereof,and the like. The locus can also include water absorbent materials suchas infected linens or other textiles. The locus also includes harvestedplants or plant products including seeds, corms, tubers, fruit, andvegetables, growing plants, and especially crop growing plants,including cereals, leaf vegetables and salad crops, root vegetables,legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, wood pulp, paper, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used toprovide a benefit to an article of clothing or a textile including, butnot limited to disinfecting, sanitizing, bleaching, destaining, anddeodorizing. The peroxycarboxylic acids formed by the present processmay be used in any number of laundry care products including, but notlimited to textile pre-wash treatments, laundry detergents, laundrydetergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents, to name a few.

The peroxycarboxylic acids formed by the present process can be used inone or more steps of the wood pulp or paper pulpbleaching/delignification process, particularly where peracetic acid isused (for example, see EP1040222 B1 and U.S. Pat. No. 5,552,018 toDevenyns, J.)

Recombinant Microbial Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the instant genesand nucleic acid molecules are microbial hosts that can be found withinthe fungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to, bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida,Hansenula, Yarrowia, Salmonella, Bacillus Acinetobacter, Zymomonas,Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus,Methanobacterium, Klebsiella, and Myxococcus. In one embodiment,bacterial host strains include Escherichia, Bacillus, and Pseudomonas.In a preferred embodiment, the bacterial host cell is Escherichia coli.

Industrial Production

A variety of culture methodologies may be applied to produce theperhydrolase catalyst. Large-scale production of a specific gene productoverexpressed from a recombinant microbial host may be produced bybatch, fed-batch or continuous culture methodologies. Batch andfed-batch culturing methods are common and well known in the art andexamples may be found in Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989) and Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992).

In one embodiment, commercial production of the desired perhydrolasecatalyst is accomplished with a continuous culture. Continuous culturesare an open system where a defined culture media is added continuouslyto a bioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Recovery of the desired perhydrolase catalysts from a batch or fed-batchfermentation, or continuous culture may be accomplished by any of themethods that are known to those skilled in the art. For example, whenthe enzyme catalyst is produced intracellularly, the cell paste isseparated from the culture medium by centrifugation or membranefiltration, optionally washed with water or an aqueous buffer at adesired pH, then a suspension of the cell paste in an aqueous buffer ata desired pH is homogenized to produce a cell extract containing thedesired enzyme catalyst. The cell extract may optionally be filteredthrough an appropriate filter aid such as celite or silica to removecell debris prior to a heat-treatment step to precipitate undesiredprotein from the enzyme catalyst solution. The solution containing thedesired enzyme catalyst may then be separated from the precipitated celldebris and protein produced during the heat-treatment step by membranefiltration or centrifugation, and the resulting partially-purifiedenzyme catalyst solution concentrated by additional membrane filtration,then optionally mixed with an appropriate excipient (for example,maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol, phosphatebuffer, citrate buffer, or mixtures thereof) and spray-dried to producea solid powder comprising the desired enzyme catalyst. Alternatively,the resulting partially-purified enzyme catalyst solution prepared asdescribed above may be optionally concentrated by additional membranefiltration, and the partially-purified enzyme catalyst solution orresulting enzyme concentrate is then optionally mixed with one or morestabilizing agents (e.g., glycerol, sorbitol, propylene glycol,1,3-propanediol, polyols, polymeric polyols, polyvinylalcohol), one ormore salts (e.g., sodium chloride, sodium sulfate, potassium chloride,potassium sulfate, or mixtures thereof), and one or more biocides, andmaintained as an aqueous solution until used.

When an amount, concentration, or other value or parameter is giveneither as a range, preferred range, or a list of upper preferable valuesand lower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limit orpreferred value and any lower range limit or preferred value, regardlessof whether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate preferredembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the methods disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the presently disclosed methods.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “μL”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “pig” means microgram(s), “ng” meansnanogram(s), “g” means gravity, “HPLC” means high performance liquidchromatography, “dd H₂O” means distilled and deionized water, “dcw”means dry cell weight, “ATCC” or “ATCC®” means the American Type CultureCollection (Manassas, Va.), “U” means unit(s) of perhydrolase activity,“rpm” means revolution(s) per minute, and “EDTA” meansethylenediaminetetraacetic acid.

Example 1 Construction of a Random Mutagenesis Library of Thermotogamaritima Acetyl Xylan Esterase C277S Variant

The coding sequence of a Thermotoga maritima acetyl xylan esterase(GENBANK® accession # NP_(—)227893.1) was synthesized using codonsoptimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.), andcloned into pUC19 between Pst1 and Xba1 to create the plasmid known aspSW202 (U.S. Patent Application Publication 2008-0176299). Thecodon-optimized sequence is provided as SEQ ID NO:1 encoding thewild-type T. maritima acetyl xylan esterase provided as SEQ ID NO: 2.

A codon change was made in the gene using primer pairs identified as SEQID NO: 3 and SEQ ID NO: 4, and the QUIKCHANGE® site-directed mutagenesiskit (Stratagene, La Jolla, Calif.), according to the manufacturer'sinstructions, resulting in the amino acid change C277S (SEQ ID NO: 5),to create the plasmid known as pSW202/C277S (SEQ ID NO: 6). PlasmidpSW202/C277S served as a template for error-prone PCR using primersidentified as SEQ ID NO: 7 and SEQ ID NO: 8, and the GENEMORPH° IIrandom mutagenesis kit (Stratagene), according to the manufacturer'srecommendations. The resulting PCR product was digested with Pst1 andXba1 and ligated with pUC19 also digested with Pst1 and Xba1. E. coliKLP18 (see U.S. 2008-0176299; herein incorporated by reference in itsentirety) was transformed with the ligation mixture and plated onto LBplates supplemented with 0.1 mg ampicillin/mL. Nucleotide sequencing ofa random sample indicated a mutation frequency of 2-8 changes per PCRproduct.

Example 2 Screening of Thermotoga Maritima Error-prone PCR Library forIncreased Enzyme Activity

Colonies were picked (automated) and placed into 96-well “master plates”containing 0.1 mL LB media supplemented with 0.1 mg ampicillin/mL andgrown 16-18 h at 37° C. and 80% humidity. From each well of the masterplates, 0.003 mL of culture was transferred to 96-well “inductionplates” containing 0.3 mL LB media supplemented with 0.1 mgampicillin/mL and 0.5 mM IPTG, which were incubated for 16-18 h withshaking at 37° C. and 80% humidity. Separately, 0.1 mL of 50% glycerolwas added to each well of the master plates, which were stored at −80°C. as stocks. From each well of the induction plates, 0.01 mL of culturewas transferred to 96-well “lysis plates” containing 0.09 mL of 56 mg/mLCELLYTIC™ Express (Sigma Aldrich, St. Louis, Mo.), which were incubatedfor 30 minutes at 30° C. From each well of the lysis plates, 0.01 mL ofmaterial was transferred to 96-well “assay plates” containing 0.045 mL“assay solution part 1” (50 mM triacetin, 50 mM potassium phosphatebuffer, pH 7.0). To each well of the assay plates was then added 0.045mL of “assay solution part 2” (50 mM hydrogen peroxide). Plates weregently mixed and incubated for 10 minutes at 30° C. To each well of theassay plate was added 0.1 mL of “stop buffer” (100 mM o-phenylenediamineand 500 mM sodium dihydrogen phosphate, pH 2.0). The plates wereincubated for 30 minutes at 30° C., after which absorbance at 458 nm wasread. T. maritima WT (codon optimized gene (SEQ ID NO:1) encoding thewild type enzyme (SEQ ID NO:2) and T. maritima C277S (SEQ ID NO:5) wereincorporated into each plate as controls. Screening of approximately5000 colonies resulted in the identification of one variant (labeled as“A3”) demonstrating activity approximately 3-fold greater than T.maritima C277S (SEQ ID NO:5). Nucleotide sequencing indicated 6additional amino acid changes in this A3 variant (F24I, S35T, Q179L,N275D, S308G, and F317S) when compared to the T. maritima acetyl xylanesterase C277S variant (SEQ ID NO:5). The “A3” strain will also bereferred to herein as the “F241/S35T/Q179L/N275D/C277S/S308G/F317Svariant”. The nucleic acid sequence of the A3 variant is provided as SEQID NO: 9 and the corresponding amino acid sequence of the A3 variant isprovided as SEQ ID NO: 10.

Example 3 Production of F2411S35T/Q179L/N275D/C277S/S308G/F3175 Variantof Thermotoga maritima Perhydrolase

Strain KLP18/pSW202/F241/S35T/Q179L/N275D/C277S/S308G/F317S was grown inLB media at 37° C. with shaking up to OD_(600nm)=0.4-0.5, at which timeIPTG was added to a final concentration of 1 mM, and incubationcontinued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGEwas performed to confirm expression of the perhydrolase enzyme at 20-40%of total soluble protein.

Example 4 Construction of N275D/C277S Variant of Thermotoga maritimaPerhydrolase

Using plasmid pSW202/C277S as starting template (see Example 1), theN275D mutation was added using the primer pair identified as SEQ ID NOs:11 and 12, and QUIKCHANGE® (Stratagene) according to the manufacturer'sinstructions. Mutations were confirmed by nucleotide sequencing, and theplasmid (pSW202/N275D/C277S) was transformed into E. coli KLP18 togenerate the strain KLP18/pSW202/N275D/C277S. The nucleic acid sequenceencoding N275D/C277S is provided as SEQ ID NO: 13. The amino acidsequence of the N275D/C277S variant is provided as SEQ ID NO: 14.

Example 5 Production of N275D/C277S Variant of Thermotoga MaritimaPerhydrolase

Strain KLP18/pSW202/N275D/C277S was grown in LB media at 37° C. withshaking up to OD_(600nm)=0.4-0.5, at which time IPTG was added to afinal concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 6 Construction of C277S/F317S Variant of Thermotoga MaritimaPerhydrolase

Using plasmid pSW202/C277S as starting template, the F317S mutation wasadded using the primer pair identified as SEQ ID NOs: 15 and 16, andQUIKCHANGE® (Stratagene) according to the manufacturer's instructions.Mutations were confirmed by nucleotide sequencing, and the plasmid(pSW202/C277S/F317S) was transformed into E. coli KLP18 to generate thestrain KLP18/pSW202/C277S/F317S. The nucleic acid sequence encodingC277S/F317S is provided as SEQ ID NO: 17. The amino acid sequence of theC277S/F317S variant is provided as SEQ ID NO: 18.

Example 7 Production of C277S/F317S Variant of Thermotoga MaritimaPerhydrolase

Strain KLP18/pSW202/C277S/F317S was grown in LB media at 37° C. withshaking up to OD_(600nm)=0.4-0.5, at which time IPTG was added to afinal concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 8 Construction of S35T/C277S Variant of Thermotoga MaritimaPerhydrolase

Using plasmid pSW202/C277S as starting template, the S35T mutation wasadded using the primer pair identified as SEQ ID NOs: 19 and 20, andQUIKCHANGE® (Stratagene) according to the manufacturer's instructions.Mutations were confirmed by nucleotide sequencing, and the plasmid(pSW202/S35T/C277S) was transformed into E. coli KLP18 to generate thestrain KLP181pSW202/S35T/C277S. The nucleic acid sequence encodingS35T/C277S is provided as SEQ ID NO: 21. The amino acid sequence of theS25T/C277S variant is provided as SEQ ID NO: 22.

Example 9 Production of S35T/C277S Variant of Thermotoga MaritimaPerhydrolase

Strain KLP18/pSW202/S35T/C277S was grown in LB media at 37° C. withshaking up to OD₆₀₀=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 10 Construction of Q179L/C277S Variant of Thermotoga MaritimaPerhydrolase

Using plasmid pSW202/C277S as starting template, the Q179L mutation wasadded using the primer pair identified as SEQ ID NOs: 23 and 24, andQUIKCHANGE® (Stratagene) according to the manufacturer's instructions.Mutations were confirmed by nucleotide sequencing, and the plasmid(pSW202/Q179L/C277S) was transformed into E. coli KLP18 to generate thestrain KLP18/pSW202/Q179L/C277S. The nucleic acid sequence encodingQ179L/C277S is provided as SEQ ID NO: 25. The amino acid sequence of theQ179L/C277S variant is provided as SEQ ID NO: 26.

Example 11 Production of Q179L/C277S Variant of Thermotoga MaritimaPerhydrolase

Strain KLP18/pSW202/Q179L/C277S was grown in LB media at 37° C. withshaking up to OD_(600nm)=0.4-0.5, at which time IPTG was added to afinal concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 12 Preparation of Cell Lysates Containing Semi-PurifiedThermotoga Maritima Variant Acetyl Xylan Esterases

Cell cultures of E. coliKLP18/pSW202/F241/S35T/Q179L/N275D/C277S/S308G/F317S,KLP18/pSW202/N275D/C277S, KLP18/pSW202/C277S/F317S,KLP18/pSW202/S35T/C277S, and KLP18/pSW202/Q179L/C277S were each grown asdescribed in Examples 3, 5, 7, 9 and 11, respectively. The resultingcell pastes were re-suspended (20% w/v) in 50 mM phosphate buffer (pH7.0) supplemented with 1.0 mM dithiothreitol (DTT). Re-suspended cellswere passed through a French pressure cell twice to ensure >95% celllysis. Lysed cells were centrifuged for 30 minutes at 12,000×g, and theresulting supernatant was heated at 75° C. for 20 minutes, followed byquenching in an ice bath for 2 minutes. Precipitated protein was removedby centrifugation for 10 minutes at 11,000×g. SOS-PAGE indicated thateach CE-7 perhydrolase variant comprised approximately 85-90% of thetotal protein in the heat-treated extract supernatant.

Example 13 Perhydrolase Specific Activity of Thermotoga MaritimaWild-Type and Variant Acetyl Xylan Esterases

Reactions (10-mL total volume) to measure perhydrolase specific activitywere run at 25° C. in phosphate buffer (50 mM, pH 7.2) containingtriacetin (100 mM), hydrogen peroxide (100 mM) and one of the followingacetyl xylan esterase variants: T. maritimaF241/S35T/Q179L/N275D/C277S/S308G/F317S perhydrolase (SEQ ID NO:10) (2.5μg/mL of heat-treated extract supernatant total protein from E. coliKLP18/pSW202/F241/S35T/Q179L/N275D/C277S/S308G/F317S), T. maritimaN275D/C277S perhydrolase (SEQ ID NO:14) (2.5 μg/mL of heat-treatedextract supernatant total protein from E. coliKLP18/pSW202/N275D/C277S), T. maritima C277S/F317S perhydrolase (SEQ IDNO:18) (2.5 μg/mL of heat-treated extract supernatant total protein fromE. coli KLP18/pSW202/C277S/F317S), T. maritima S35T/C277S perhydrolase(SEQ ID NO:22) (2.5 μg/mL of heat-treated extract supernatant totalprotein from E. coli KLP18/pSW202/S35T/C277S), T. maritima Q179L/C277Sperhydrolase (SEQ ID NO:26) (2.5 μg/mL of heat-treated extractsupernatant total protein from E. coli KLP18/pSW202/Q179L/C277S) (allprepared as described in Example 12). Reactions were stirred for onlythe first 30 seconds of reaction to initially mix the reactants andenzyme. A sample from each of the reaction mixtures described above waswithdrawn after the first minute of each reaction, and every two minutesthereafter for fifteen minutes, and each sample was analyzed forperacetic acid by reaction with methyl-p-tolyl sulfide (MTS, see below).

Measurement of the rate of peracetic acid production in the reactionmixture was performed using a modification of the method described byKarst et al. (Anal. Chem., 69(17):3623-3627 (1997)). A sample (0.040 mL)of the reaction mixture was removed at predetermined times as describedabove and immediately mixed with 0.960 mL of 5 mM phosphoric acid inwater to terminate the reaction by adjusting the pH of the dilutedsample to between pH 3 and pH 4. The resulting solution was filteredusing an ULTRAFREE® MC-filter unit (30,000 Normal Molecular Weight Limit(NMWL), Millipore Corp., Billerica, Mass.; cat #UFC3LKT 00) bycentrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of theresulting filtrate was transferred to a 1.5-mL screw cap HPLC vial(Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing 0.300mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-tolylsulfide) in acetonitrile was added, the vial capped, and the contentsbriefly mixed prior to a 10 min incubation at ca. 25° C. in the absenceof light. To the vial was then added 0.400 mL of acetonitrile and 0.100mL of a solution of triphenylphosphine (TPP, 40 mM) in acetonitrile, thevial re-capped, and the resulting solution mixed and incubated at ca.25° C. for 30 min in the absence of light. To the vial was then added0.100 mL of 10 mM N,N-diethyl-m-toluamide (DEET; HPLC external standard)and the resulting solution analyzed by HPLC for MTSO (methyl-p-tolylsulfoxide), the stoichiometric oxidation product produced by reaction ofMTS with peracetic acid. A control reaction was run in the absence ofadded extract protein or triacetin to determine the rate of oxidation ofMTS in the assay mixture by hydrogen peroxide, for correction of therate of peracetic acid production for background MTS oxidation.

HPLC method: Supelco Discovery C8 column (10-cm×4.0-mm, 5 μm) (catalog#569422-U) with Supelco Supelguard Discovery C8 precolumn (Supelco;catalog #59590-U); 10 microliter injection volume; gradient method withCH₃CN (Sigma-Aldrich; catalog #270717) and deionized water at to mL/minand ambient temperature (Table 4).

TABLE 4 HPLC Gradient for analysis of peracetic acid. Time (min:sec) (%CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00 (stop) 40

Reactions were also run under identical conditions to that describedimmediately above using either T. maritima wild-type acetyl xylanesterase (SEQ ID NO:2) (50 μg/mL of heat-treated extract supernatanttotal protein from E. coli KLP18/pSW202) or T. maritima C277S variantacetyl xylan esterase (SEQ ID NO:5) (2.5 μg/mL of heat-treated extractsupernatant total protein from E. coli KLP18/pSW202/C277S) (see U.S.Patent Application Publication 2008-0176299 and U.S. patent applicationSer. No. 12/572,094), where the heat-treated extract supernatant wasprepared according to the procedure of Example 12. The perhydrolysisreaction rate and the perhydrolase specific activity for perhydrolysisof triacetin to peracetic acid for each of the perhydrolases arereported in Table 5.

TABLE 5 Perhydrolase specific activity (PAAF specific activity) forThermotoga maritima wild-type and variant perhydrolases. perhydrolaseThermotoga SEQ perhydrolase perhydrolysis specific maritima ID concen.rate activity (U/ perhydrolase NO: (μg/mL) (mM/min) mg protein) wildtype 2 50 5.36 107 C277S 5 2.5 1.97 788 N275D/C277S 14 2.5 2.03 812C277S/F317S 18 2.5 2.09 836 S35T/C277S 22 2.5 1.07 428 Q179L/C277S 262.5 1.52 608 F24I/S35T/Q179L/ 10 2.5 2.77 1108 N275D/C277S/ S308G/F317S

Example 14 Peracid Hydrolysis Specific Activity of Thermotoga MaritimaWild-Type and Variant Acetyl Xylan Esterases

Reactions (10-mL total volume) to measure peracid hydrolysis specificactivity were run at 25° C. in phosphate buffer (50 mM, pH 7.2)containing peracetic acid (26.3 mM, 2000 ppm) and one of the followingacetyl xylan esterase variants: T. maritimaF241/S35T/Q179L/N275D/C277S/S308G/F317S perhydrolase (SEQ ID NO:10) (10μg/mL of heat-treated extract supernatant total protein from E. coliKLP18/pSW202/F241/S35T/Q179L/N275D/C277S1S308G/F317S), T. maritimaN275D/C277S perhydrolase (SEQ ID NO:14) (25 μg/mL of heat-treatedextract supernatant total protein from E. coliKLP18/pSW202/N275D/C277S), T. maritima C277S/F317S perhydrolase (SEQ IDNO:18) (25 μg/mL of heat-treated extract supernatant total protein fromE. coli KLP18/pSW202/C277S/F317S), T. maritima S35T/C277S perhydrolase(SEQ ID NO:22) (25 μg/mL of heat-treated extract supernatant totalprotein from E. coli KLP18/pSW202/S35T/C277S), T. maritima Q179L/C277Sperhydrolase (SEQ ID NO:26) (25 μg/mL of heat-treated extractsupernatant total protein from E. coli KLP18/pSW202/Q1791/C277S (allprepared as described in Example 12). Reactions were stirred for onlythe first 30 seconds of reaction to initially mix the reactants andenzyme. A sample from each of the reaction mixtures described above waswithdrawn after the first minute of each reaction, and every two minutesthereafter for fifteen minutes, and each sample was analyzed forperacetic acid using a modification of the method described by Karst etal., supra.

Reactions were also run under identical conditions to that describedimmediately above using either T. maritima wild-type perhydrolase (SEQID NO:2) (50 μg/mL of heat-treated extract supernatant total proteinfrom E. coli KLP18/pSW202) or T. maritima C2773 variant perhydrolase(SEQ ID NO:5) (10 μg/mL of heat-treated extract supernatant totalprotein from E. coli KLP18/pSW202/C277S), where the heat-treated extractsupernatant was prepared according to the procedure of Example 12. Theperacid hydrolysis reaction rate and the peracid hydrolysis specificactivity for hydrolysis of peracetic acid to acetic acid and hydrogenperoxide for each perhydrolase are reported in Table 6.

TABLE 6 Peracid hydrolysis specific activity (PAAH specific activity) ofThermotoga maritima wild-type and variant perhydrolases. peracid peracidhydrolysis Thermotoga SEQ perhydrolase hydrolysis specific maritima IDconcen. rate activity (U/ perhydrolase NO: (μg/mL) (mM/min) mg protein)wild type 2 50 1.21 24.3 C277S 5 10 0.54 54.1 N275D/C277S 14 25 0.6425.6 C277S/F317S 18 25 1.08 43.2 S35T/C277S 22 25 0.53 21.2 Q179L/C277S26 25 0.60 24.0 F24I/S35T/Q179L/ 10 10 0.55 55.0 N275D/C277S/S308G/F317S

Example 15 Comparison of the Relative Perhydrolase (PAAF) and PeracidHydrolysis (PAAH) Specific Activities of Thermotoga Maritima Wild-Typeand Variant Acetyl Xylan Esterases

Table 7 reports the perhydrolase specific activity (Example 13), theperacid hydrolysis specific activity (Example 14), and the ratio ofperhydrolase specific activity to peracid hydrolysis specific activityfor each of the listed T. maritima wild-type and variant acetyl xylanesterases. The N275D/C277S (SEQ ID NO: 14),F241/S35T/Q179L/N275D/S308G/F317S/C277S (SEQ ID NO: 10), C277S/F317S(SEQ ID NO:18), S35T/C277S (SEQ ID NO:22), and Q179L/C277S (SEQ IDNO:26) acetyl xylan esterases each had an improved ratio of perhydrolasespecific activity for peracetic acid formation (PAAF) to peracetic acidhydrolysis specific activity (PAAH) when compared to either the T.maritima wild-type (SEQ ID NO:2) or the C277S variant (SEQ ID NO:5)perhydrolase.

TABLE 7 Relative Perhydrolase (PAAF) and Peracid Hydrolysis (PAAH)Specific Activities of Thermotoga maritima Wild-type and Variant AcetylXylan Esterases. ratio of perhydrolase peracid hydrolysis (perhydrolase/Fold Increase of Fold Increase of Thermotoga maritima specific activityspecific activity peracid hydrolysis) PAAF/PAAH ratio PAAF/PAAH ratioperhydrolase SEQ ID NO: (U/mg protein) (U/mg protein) specificactivities vs. Wild-Type vs. C277S Variant Wild-type 2 107 24.3 4.4 — —C277S 5 788 54.1 14.6 3.3 — N275D/C277S 14 812 25.6 31.7 7.2 2.2C277S/F317S 18 836 43.2 19.4 4.4 1.3 S35T/C277S 22 428 21.2 20.2 4.6 1.4Q179L/C277S 26 608 24.0 25.3 5.8 1.7 F24I/S35T/Q179L/N275D/ 10 1108 55.020.1 4.6 1.4 C277S/S308G/F317S

1. An isolated polypeptide having perhydrolytic activity comprising theamino acid sequence of SEQ ID NO:
 10. 2. The polypeptide of claim 1;wherein said polypeptide is characterized by a peracetic acid formationspecific activity (PAAF) to peracetic acid hydrolysis specific activity(PAAH) ratio (PAAF/PAAH) that is at least 1.1-fold higher than thePAAF/PAAH ratio of the Thermotoga maritima acetyl xylan esterase havingamino acid sequence SEQ ID NO:
 2. 3. A process for producing aperoxycarboxylic acid comprising: (a) providing a set of reactioncomponents comprising: (1) at least one substrate selected from thegroup consisting of: (i) one or more esters having the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆=C2 to C7; R₅=C_(y) linear,branched, or cyclic hydrocarbyl moiety optionally substituted withhydroxyl groups; wherein each carbon atom in R₅ individually comprisesno more than one hydroxyl group or no more than one ester group; whereinR₅ optionally comprises one or more ether linkages; y=1 to 6; m=1 to 6,provided that m≦y; and wherein said esters have solubility in water ofat least 5 ppm at 25° C.; (ii) one or more glycerides having thestructure

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acetylatedmonosaccharides, acetylated disaccharides, or acetylatedpolysaccharides; and (v) any combination of (i) through (iv); (2) asource of peroxygen; and (3) an enzyme catalyst comprising thepolypeptide of claim 1; (b) combining the set of reaction componentsunder suitable reaction conditions whereby peroxycarboxylic acid isproduced; and (c) optionally diluting the peroxycarboxylic acid producedin step (b).
 4. The process of claim 3 further comprising the step of:d) contacting a hard surface or inanimate object with theperoxycarboxylic acid produced in step (b) or step (c); whereby saidhard surface or said inanimate object is disinfected, bleached,destained or a combination thereof.
 5. The process of claim 3 whereinthe inanimate object is a medical instrument.
 6. The process of claim 3further comprising the step of: d) contacting an article of clothing ora textile with peroxycarboxylic acid produced in step (b) or step (c).7. The process of claim 6 wherein the article of clothing or the textileis disinfected, sanitized, bleached, destained, deodorized, or acombination thereof.
 8. The process of claim 3 further comprising thestep of: d) contacting wood pulp or paper pulp with peroxycarboxylicacid produced in step (b) or step (c); whereby the wood pulp or paperpulp is bleached.
 9. The process of claim 3 wherein the substrate isselected from the group consisting of: monoacetin; diacetin; triacetin;monopropionin; dipropionin; tripropionin; monobutyrin; dibutyrin;tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan;acetylated xylan fragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanedial,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; and mixtures thereof.
 10. The process of claim 9 whereinthe substrate is triacetin.
 11. The process of claim 3 wherein theperoxycarboxylic acid produced is peracetic acid, perpropionic acid,perbutyric acid, perlactic acid, perglycolic acid, permethoxyaceticacid, per-β-hydroxybutyric acid, or mixtures thereof.
 12. The process ofclaim 3 wherein the enzyme catalyst is in the form of a microbial cell,a permeabilized microbial cell, a microbial cell extract, a partiallypurified enzyme, or a purified enzyme.
 13. A composition comprising: (a)a set of reaction components comprising: (1) at least one substrateselected from the group consisting of: one or more esters having thestructure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆=C2 to C7; R₅=C_(y) linear,branched, or cyclic hydrocarbyl moiety optionally substituted withhydroxyl groups; wherein each carbon atom in R₅ individually comprisesno more than one hydroxyl group or no more than one ester group; whereinR₅ optionally comprises one or more ether linkages; y=1 to 6; m=1 to 6,provided that m≦y; and wherein said esters have solubility in water ofat least 5 ppm at 25° C.; (ii) one or more glycerides having thestructure

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acetylatedmonosaccharides, acetylated disaccharides, or acetylatedpolysaccharides; and (v) any combination of (i) through (iv); (2) asource of peroxygen; and (3) an enzyme catalyst comprising thepolypeptide of claim 1; and (b) at least one peroxycarboxylic acidformed upon combining the set of reaction components of (a).
 14. Aperacid generation and delivery system comprising: (a) a firstcompartment comprising (1) an enzyme catalyst comprising the polypeptideof claim 1; (2) at least one substrate selected from the groupconsisting of: (i) one or more esters having the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆=C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆=C2 to C7; R₅=C_(y) linear,branched, or cyclic hydrocarbyl moiety optionally substituted withhydroxyl groups; wherein each carbon atom in R₅ individually comprisesno more than one hydroxyl group or no more than one ester group; whereinR₅ optionally comprises one or more ether linkages; y=1 to 6; m=1 to 6,provided that m≦y; and wherein said esters have solubility in water ofat least 5 ppm at 25° C.; (ii) one or more glycerides having thestructure

wherein R₁=C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acetylatedmonosaccharides, acetylated disaccharides, or acetylatedpolysaccharides; and (v) any combination of (i) through (iv); and (3) anoptional buffer; and (b) a second compartment comprising (1) source ofperoxygen; (2) a peroxide stabilizer; and (3) an optional buffer. 15.The peracid generation and delivery system of claim 14 wherein thesubstrate comprises triacetin.
 16. A laundry care product comprising thepolypeptide of claim 1.